Ivan Brukner

Host and Pathogen Factors for Clostridium difficile Infection and Colonization

BACKGROUND Clostridium difficile infection is the leading cause of health care-associated diarrhea, and the bacterium can also be carried asymptomatically. The objective of this study was to identify host and bacterial factors associated with health care-associated acquisition of C. difficile infection and colonization. METHODS We conducted a 15-month prospective study in six Canadian hospitals in Quebec and Ontario. Demographic information, known risk factors, potential confounding factors, and weekly stool samples or rectal swabs were collected. Pulsed-field gel electrophoresis (PFGE) was performed on C. difficile isolates to determine the genotype. Levels of serum antibodies against C. difficile toxins A and B were measured. RESULTS A total of 4143 patients were included in the study; 117 (2.8%) and 123 (3.0%) had health care-associated C. difficile infection and colonization, respectively. Older age and use of antibiotics and proton-pump inhibitors were significantly associated with health care-associated C. difficile infection. Hospitalization in the previous 2 months; use of chemotherapy, proton-pump inhibitors, and H(2) blockers; and antibodies against toxin B were associated with health care-associated C. difficile colonization. Among patients with health care-associated C. difficile infection and those with colonization, 62.7% and 36.1%, respectively, had the North American PFGE type 1 (NAP1) strain. CONCLUSIONS In this study, health care-associated C. difficile infection and colonization were differentially associated with defined host and pathogen variables. The NAP1 strain was predominant among patients with C. difficile infection, whereas asymptomatic patients were more likely to be colonized with other strains. (Funded by the Consortium de Recherche sur le Clostridium difficile.).

Fourteen-Genome Comparison Identifies DNA Markers for Severe-Disease-Associated Strains of Clostridium difficile

ABSTRACT Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ∼3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.

DNA Variants in Region for Noncoding Interfering Transcript of Dihydrofolate Reductase Gene and Outcome in Childhood Acute Lymphoblastic Leukemia

Purpose: Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event-free survival correlated with haplotype *1, defined by A317 and C1610 alleles. Haplotype *1 was also associated higher DHFR expression. Experimental Design: Here, we analyzed adjacent 400-bp region participating in DHFR regulation as both a major promoter and a noncoding minor transcript. Results: Six polymorphisms were identified, of which five were single nucleotide polymorphisms and one was length polymorphism composed of variable number of 9-bp elements and 9-bp insertion/deletion. Haplotype analysis including all promoter polymorphisms revealed diversification of haplotype *1 into five subtypes (*1a-*1e). DNA variations of major promoter/noncoding transcript region and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower event-free survival was associated with an A allele of G308A polymorphism (P = 0.02) and with *1b haplotype (P = 0.01). This association was particularly striking in high-risk patients (P = 0.001) and was subsequently confirmed in independent patient cohort (P = 0.02). Haplotype *1b was the only haplotype *1 subtype associated with higher mRNA levels. Conclusions: The study provides a new insight into DHFR regulatory variations predisposing to an event in ALL patients. (Clin Cancer Res 2009;15(22):69318)

Bim Polymorphisms: Influence on Function and Response to Treatment in Children with Acute Lymphoblastic Leukemia

Purpose: Corticosteroids induce apoptosis in the malignant lymphoid cells and are critical component of combination therapy for acute lymphoblastic leukemia (ALL). Several genome-wide microarray studies showed major implication of proapoptotic Bim in mediating corticosteroid-related resistance in leukemia cells. Experimental Design: We investigated Bim gene polymorphisms and their association with childhood ALL outcome, and the mechanism underlying the observed finding. Results: Lower overall survival (OS) was associated with Bim C29201T located in Bcl-2 homology 3 (BH3) domain (P = 0.01). An association remained significant in multivariate model (P = 0.007), was more apparent in high-risk patients (P = 0.004) and patients treated with dexamethasone (P = 0.009), and was subsequently confirmed in the replication patient cohort (P = 0.03). RNA analysis revealed that C29201T affects generation of γ isoforms (γ1) that lack proapoptotic BH3 domain. The phenotypic effect was minor suggesting the influence of additional factors that may act in conjunction with Bim genotype. Combined analysis with Mcl gene polymorphism (G-486T) revealed profound reduction in OS in individuals with both risk genotypes (P < 0.0005 in discovery and P = 0.002 in replication cohort) and particularly in high-risk patients (P ≤ 0.008). Conclusions: Increased expression of prosurvival Mcl1 and presence of Bim isoforms lacking proapoptotic function might explain marked reduction of OS in a disease and dose-dependent manner in ALL patients carrying Bim- and Mcl1-risk genotypes. Clin Cancer Res; 19(18); 5240–9. ©2013 AACR.

Newly-isolated HPV97, related to HPV18 and 45 is frequently detected in HIV-positive men from the Montreal area

This may reflect that themost prevalent types were isolated and characterized earlier,and not many mucosal types are left to discover. Recentlycloned types 92, 93, 94, 95 and 96 were isolated from skinlesions and belong to the cutaneous Beta Papillomavirus genuswhich has a less important influence on human health.

A protocol for the in vitro selection of specific oligonucleotide probes for high-resolution DNA typing

The confident discrimination of nucleic acids that share a high degree of sequence identity is the major obstacle for the widespread applicability of multiplex DNA-based techniques. This diagnostic uncertainty originates in the insufficient specificity of hybridization, allowing cross-hybridization between unwanted probe–target combinations. Starting from a random mixture of oligonucleotides, we describe a protocol to selectively amplify the probes that bind to the target but not to the similar, unintended targets. The procedure involves five forward hybridizations to generate pools of probes with significant affinity, but not necessarily specificity, for the target. Specificity is then achieved during subtractive hybridization steps, where only probes having differential diagnostic performance are retained. Iterative hybridizations, cloning, sequencing and testing of the performance of selected probes can all be fully automated. Eight weeks are required for the full completion of a project composed of 40 probe–target pairs, even when targets share as much as 87% of sequence identity. While alternative, computer-assisted, rational oligonucleotide design may produce an uncertain outcome, the present protocol generates robust and specific probes suitable for a variety of multiplex, nucleic acid-based detection/typing platforms.

Characterization of the microDNA through the response to chemotherapeutics in lymphoblastoid cell lines

Recently, a new class of extrachromosomal circular DNA, called microDNA, was identified. They are on average 100 to 400 bp long and are derived from unique non-repetitive genomic regions with high gene density. MicroDNAs are thought to arise from DNA breaks associated with RNA metabolism or replication slippage. Given the paucity of information on this entirely novel phenomenon, we aimed to get an additional insight into microDNA features by performing the microDNA analysis in 20 independent human lymphoblastoid cell lines (LCLs) prior and after treatment with chemotherapeutic drugs. The results showed non-random genesis of microDNA clusters from the active regions of the genome. The size periodicity of 190 bp was observed, which matches DNA fragmentation typical for apoptotic cells. The chemotherapeutic drug-induced apoptosis of LCLs increased both number and size of clusters further suggesting that part of microDNAs could result from the programmed cell death. Interestingly, proportion of identified microDNA sequences has common loci of origin when compared between cell line experiments. While compatible with the original observation that microDNAs originate from a normal physiological process, obtained results imply complementary source of its production. Furthermore, non-random genesis of microDNAs depicted by redundancy between samples makes these entities possible candidates for new biomarker generation.

Detection and Isolation of Clostridium difficile Asymptomatic Carriers During Clostridium difficile Infection Outbreaks: An Exploratory Study

During 4 Clostridium difficile infection outbreaks, unit-wide screening of 114 patients led to detection and isolation of 15 (13%) C. difficile asymptomatic carriers. Carriage prevalence varied between outbreaks, from 0% to 29% (P = .004). Isolating carriers was not associated with significantly shorter outbreak durations, compared with historical controls.